1. Technical Field
The present invention relates to a time-resolved fluorescence microscope capable of carrying out a femtosecond-order time-resolved measurement of fluorescence from a microregion.
2. Background Art
A time-resolved fluorescence microscope utilizing time-resolved fluorescence spectroscopy and an optical microscope in combination is a useful apparatus that can provide information relating to the electronic states of molecules in a microregion accurately without interference from other factors. Time-resolved techniques used in the conventional time-resolved fluorescence microscope can be roughly classified into two groups. One is the time-correlated single-photon counting method, and the other is the streak camera method.
FIG. 2 shows an example of a time-correlated single-photon counting apparatus (see Non-patent document 1). Pulsed light emitted by a laser diode 51 passes through a filter 52 and is then condensed by a microscope objective lens (oil-immersion objective) 53 to produce an excitation light, with which a microregion on a sample 54 is photoexcited. The excitation-light irradiated position on the sample can be adjusted by finely moving the sample while monitoring an enlarged sample image using a monitor 56 captured by a CCD camera 55. Fluorescence emitted by the sample due to irradiation with the excitation light is subjected to wavelength selection in filters 57 and 58, and then detected by a photodiode 60 via a pin hole 59. The resultant electronic signal pulse from the photodiode 60 is fed to a time-to-amplitude converter (TAC) 61. On the other hand, an electric pulse from laser diode is fed to TAC 61 as a trigger. TAC 61 outputs an electric pulse corresponding to the time difference between the trigger pulse and a signal pulse from a photodiode 60. The electric pulse is then fed to a multi-channel analyzer (MCA) 62 to obtain the distribution of time difference and number of signals from a photodiode. As a result, a waveform can be obtained that corresponds to the time decay of the fluorescence from the sample disposed under the microscope.
The streak camera method involves the introduction of fluorescence from a sample under a microscope into a streak tube after passing through a spectroscope, using an electric signal from laser as a trigger. The incident light hits the photo-cathode, whereby the light is converted into electrons. The electrons thus produced are accelerated, and their flying direction is changed by a voltage applied between the top and bottom of the streak tube. By varying the applied voltage at high speed, the time distribution of fluorescence can be captured in terms of spatial changes (see Non-patent document 2).
As a method of time-resolved measurement of fluorescence emitted by a bulk sample, a sum-frequency generation method (up-conversion method) is known, whereby a pulsed light emitted by laser is divided in two. One portion of the pulsed light is used as a sample excitation light, and fluorescence emitted by the sample is mixed with the other portion of the pulsed light (gate pulse) in a non-linear optical crystal. Then, the light of the sum frequency of the mixed light emitted by the non-linear optical crystal is detected (see Non-patent document 3). In this method, time-resolved measurement of fluorescence can be carried out by repeating the measurement while varying the delay time of the gate pulse.
Non-patent document 1: Review of Scientific Instruments, vol. 70, pp.1835-1841, 1999
Non-patent document 2: Applied Physics Letters, vol. 80, No. 18, pp.3340-3342, 2002
Non-patent document 3: Journal of Physical Chemistry A, vol. 101, pp.3052-3060, 1997
Generally, in order to analyze the various dynamic behaviors (chemical reactions, energy transfer, etc.) of objects, time resolution of the order of sub-picoseconds (10−12 seconds or less) to several hundred femtoseconds (10−13 seconds) is required. However, the conventional time-resolved fluorescence microscopes can only provide insufficient time resolution of the order of nanosecond (10−9 seconds) to several tens of picoseconds (10−11 seconds). This is due to the fact that they employ methods based on a single-photon counting apparatus or a streak camera for time-resolved measurement, by which fluorescence is electrically processed. In fact, time resolution of the order of 40 ps is the limit in apparatuses employing the time-correlated single-photon counting method. Further, the time resolution of time-resolved fluorescence microscopes using a streak tube is of the order of 5 ps at most. The streak camera method can be used for time-resolved measurement without the use of a spectroscope, whereby high time resolution of the order of 300 femtoseconds can be obtained. In this case, however, the wavelength of fluorescence cannot be determined. While the up-conversion method is capable of measuring fluorescence with high time resolution, it is not adapted for the measurement of microregions and cannot be expected to provide microscopic spatial resolution as is.
A confocal microscope, which is a kind of optical microscope, is capable of selectively measuring depthwise information about an object by reducing the pin hole size provided in the microscope. However, in actual measurements, the size of the pin hole must be sufficiently large such that a sufficiently strong signal can be obtained. Thus, the confocal microscope has a limited spatial resolution in the depth direction. Thus, it is very much desired to improve the depth resolution of the confocal microscope.